ORIGINAL ARTICLE AFRICAN JOURNAL OF CLINICAL AND EXPERIMENTAL MICROBIOLOGY. JANUARY 2014 ISBN 1595 689X VOL15 No.1 AJCEM/1322 http://www.ajol.info/journals/ajcem COPYRIGHT 2014 http://dx.doi.org/10.4314/ajcem.v15i1.1 AFR. J. CLN. EXPER. MICROBIOL 15(1): 1 8
DIVERSE GENETIC SUBTYPES OF HIV 1 AMONG FEMALE SEX WORKERS IN IBADAN, NIGERIA
*1Fayemiwo, S. A., 2Odaibo, G. N. 3 Sankale , J.L., 1Oni, A.A., 1Bakare, R. A., 2Olaleye, O. D. and 4Kanki, P.
Departments of 1Medical Microbiology and 2Virology, College of Medicine, University College Hospital, Ibadan, Nigeria and 3A.P.I.N., Harvard School of Public Health, Boston, MA , USA.
Running title: Genetic subtypes of HIV 1 among female sex workers in Nigeria.
Keywords: Diverse, HIV, subtypes, Female Sex workers and Vaccine
*Correspondence: Fayemiwo, S. A., Department of Medical Microbiology and Parasitology, College of Medicine, University of Ibadan, Ibadan. Nigeria. E mail address: [email protected]
ABSTRACT Background:
Genetic diversity is the hallmark of HIV 1 infection. It differs among geographical regions throughout the world. This study was undertaken to identify the predominant HIV 1 subtypes among infected female sex workers (FSWs) in Nigeria. Methods: Two hundred and fifty FSWs from brothels in Ibadan Nigeria were screened for HIV antibody using ELISA. All reactive samples were further tested by the Western Blot Techniques. Peripheral Blood Mononuclear Cells (PBMCs) were separated from the blood samples of each subject. Fragments of HIV Proviral DNA was amplified and genetic subtypes of HIV 1 was determined by direct sequencing of the env and gag genes of the viral genome followed by phylogenetic analysis . Results: The age of the FSWs ranged from 15 to 55 years old (Mean = 25.8years, SD =3.74). Majority were Nigerians while others (1.6 %) were from neighboring West Africa countries. Four ( 1.6% ) of the FSWs were active for less than one year as sex workers, and the mean length of sex work was 2.80 years ( Range = 1.0 – 15.0 years ). Sixty four (25.6%) of the 250 CSWs were positive for HIV 1 while 7 (2 8%) had dual infections to HIV 1 / HIV 2. Among the 34 HIV 1 strains characterized by sequencing, 19 (55 9%) were subtype G, 9 (26.5%) CRF02_A/G, 3 (8.8%) CRF06_cpx while 1 (2.9%) each were identified as subtype C, CRF01_A/E and CRF09_cpx respectively. Nineteen (55.9%) of the FSWs with subtype G had been active in the sex work for between one to five years. The youngest of the HIV 1 infected FSWs with sexual activity of less than a year had subtype G strain. There is a significant probability that infection with this subtype occurred with a short incubation period (p< 0.05). Conclusion: This study showed a wide range of HIV 1 subtypes among FSWs in Nigeria. The situation poses serious challenge for the design of HIV vaccine candidate for use in Nigeria.
INTRODUCTION Female sex workers (FSWs) have been at high risk of infection since the beginning of the HIV epidemic all The Acquired Immune Deficiency Syndrome (AIDS) over the world. Multiple sex partners, irregular epidemic has exacted a terrible toll in terms of loss of condom use, and co infection with other sexual life and decreased quantity of life worldwide, transmitted infections (STIs) are the principal risk especially in Africa, where 70% of deaths from HIV 1 factors for HIV infection among FSWs (7). Between infection have occurred (1). 1995 and 2003 the prevalence rose from 6.4% in STD patients (8) to 34.3% in FSWs (9). The penetration into The first case of AIDS in Nigeria was reported in the general population cannot be over emphasized. 1986. From 2 cases in 1986, it has been estimated that 4,050,000 HIV infections have occurred in Nigeria 2, In women, the glandular epithelia have HIV in the and it has spread extensively to the rural areas of the zone of transformation between the columnar and Country (3, 4, 5). squamous cell of the cervix (10). While in men, HIV is detectable in seminal cells, some sperm cells and their In Africa, infection has been mainly by heterosexual precursors (11). Because of high incidence of sexually intercourse, with Female sex workers (FSWs), long transmitted infections (STIs) in African sex workers, distant truck drivers and migrant labour serving as immunologic stimulations to this infection may be vehicles of spread (5). higher than among the general African population (12).
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HIV 1 and HIV 2 show heterogeneity among isolates MATERIALS AND METHODS (13). On the basis of the phylogenetic analysis of the env and gag genes, HIV 1 has been divided into three Study population and Sample collection distinct groups (M, O, & N.), all of which cause AIDS in infected individuals. Group M (“For Major”) A cross sectional seroprevalence study was comprises the greatest majority of HIV 1 infections conducted in a population at high risk for HIV worldwide and has been further subdivided into infection from November 2002 and July 2003 in subtypes and sub subtypes termed A D, F.H, J, K, Ibadan, South Western Nigeria. Only those subjects and Circulating Recombinants Forms; CRF 01 to 34 who were willing to participate were provided (13,14). Whereas several HIV 1 subtypes co circulate written informed consent, enrolled, and sampled. A in many parts of the world, some regions seem to total of two hundred and fifty Female sex workers have predominant subtypes. Subtype B is the most from various brothels in Ibadan that had been in the predominant form in North America and Europe profession for at least one month and duly signed while subtype E is highly prevalent in part of South informed consent were recruited into the study. The East Asia (where B is the minority form) (15). study protocol was approved by the University of Subtypes A, C, and D are the most common subtypes Ibadan/ University College Hospital (UCH) ethical in Africa. In Mali 80% of the HIV 1 infected sex review committee. Semi structured questionnaire was workers were subtype A, 10% belong to subtype G administered to each of the FSW who gave informed and the minority belong to subtypes C and D (16), consent, as an instrument to elicit base line while in Kenya 56% of the infected subjects had information on their demographic characteristics and subtypes A while others have subtypes C and D with reproductive health history. other recombinants circulating forms (17). Ten millimeters of EDTA – anti coagulated blood In Nigeria, the first HIV 1 strain that was partially samples were collected from the recruited female sex characterized was found to be subtype A (18). In workers. These samples were transported on an ice recent time , we have co circulation of multiple parks in an insulated box to the Diagnostic subtypes including, A, B, C, D, E and other groups in Laboratory, Virology Department, University College Nigeria with subtype C being the most prevalent (19). Hospital, Ibadan for processing. Plasma or sera were subsequently separated and stored in aliquots and The city of Ibadan, which is acclaimed to be the maintained at 70 oc until tested. Clinical examinations largest city in West Africa, and capital of Oyo State, is of the lower genito urinary tracts were carried out for a major commercial centre with long distant transport signs of infections such as vaginal discharge, workers, migrant labour workers, civil servants and endocervical discharge, genital ulcer diseases and students of higher institutions which have sex with genital mass (genital warts and bartholin’s cyst). multiple female sex workers with risk of acquiring HIV infection.
The occurrences of these multiple variants of HIV in Antibody Screening and Confirmation different geographical regions raise the possibility All the sera or plasma separated from the samples that future vaccine strategies may have to consider collected were screened using Enzyme Linked polyvalent constructs. The effective evaluation of Immunosorbent Assay (ELISA) techniques with vaccine candidates will depend on thorough commercial available kits (GENSCREEN PLUS HIV surveillance and characterization of viral variants by Bio Rad in France). All reactive samples were (20). confirmed by Western blot assay (New LAV BLOT 1 Since HIV infection most often results from by Bio Rad, in France). heterosexual intercourse, female sex workers in DNA Extraction and Polymerase Chain Reactions various brothels in Ibadan are likely to be the source of detection of new subtypes that might have been Qiagen kit was used for the extraction of DNA from introduced into the country and likely point for rapid the Peripheral Blood Mononuclear Cells separated spread of these different subtypes. Therefore this from the whole blood of the FSWs that had been study was aimed at providing information on the previously stored and maintained at 70 0C. The sero prevalence of HIV among female sex workers in Qiagen genomic DNA purification procedure is Ibadan and also to identify the predominant subtypes designed to isolate chromosomal DNA 20 150kb of HIV 1 among them. directly from the whole blood, buffy coat and cultured cells. Genomic DNA is eluted in a high salt
buffer, concentrated and desalted by isopropanol
2 precipitation. About 5ul of the genomic DNA of the RESULTS confirmed ELISA reactive blood samples were combined with primers: WTI (KK30) and WT2 A total of 250 Female sex workers (FSWs) from (KK40), buffers, nucleotides , taq polymerase and various brothels in Ibadan were enrolled and distilled water in a micro centrifuge tubes. Positive screened for HIV antibody seropositivity and and negative controls were included in each run. The predominant HIV 1/2 subtypes was determined total volume in each tube was made up to 50 ul in the during the period of study. As shown in Table 1, 1st round of PCR. Each tube was placed in a thermal demographic characteristics of the FSWs showed that cycler and subjected to PCR under the following their mean age was 28.8years (Range 15 55yrs; SD = cyclic conditions: 94 oc, 55 oc and 72 oc for 1 minute (5 7.35). Majority (62.8%) were in the 20 29 yrs age cycles) : 94 0C for 50s, 57 oC for 50s and 72 oC for 1mim group. 174(69.6%) were singles 37(14.8%) were 30s (30 cycles) and 72 o for 1mimute (1Cycle) divorced, 7(2.8%) were separated while 30(12.0%) were still married. The 2 nd round amplification was performed with 5ul of the 1 st round PCR products, primers (KK30, KK40), buffers, nucleotides, taq polymerase and distilled More than two third of the uninfected FSWs (71.5%) water adding up to 100ul of total volume. These were were single while 11.8 % were married, others were also subjected to the same cyclic conditions except divorced, separated or widowed. Majority (98.4%) that the five cycles initially were reduced to three. were from different parts of Nigeria while 1.6% were Detection of Amplified Products and Sequencing from Republic of Benin, Togo, Ghana and Cote Detection methods that rely on size separation and d’Ivoire. Forty one (64.1%) of the HIV – infected visualization of PCR products include agarose and FSWs had their first sexual debut between the ages of polyacrylamide gel electrophoresis(21,22). 16 20 yrs and more than half (57.8%) usually attended to one to five sexual partners per day. (Table1). These methods allow the assessment of the presence and the size of the amplified products. Agarose gel used to separate a wide range of DNA fragment sizes. Sixty four (25.6%) of the 250 FSWs screened for Visualization was achieved by staining the gel with antibody to HIV were confirmed HIV 1 seropositive ethidium bromide, a chromogen dye that binds to by the Western Blot technique. Seven (2.8%) had dual nucleic acid and florescence under UV light. infections to HIV 1/2.There was no FSW with only Genome fragments could be amplified and directly HIV 2 infection. The number of HIV 1 infection was sequenced (without cloning) using a cycle sequencing significantly higher than HIV 2 (p<0.05). Thus the protocol adapted for the ABI automatic sequencer. seroprevalence of HIV 1 antibody among the Female In order to determine the subtype of genome sex workers in Ibadan was 25.6%. fragments or entire HIV 1 genome, the sequences in questions were aligned with reference sequences of different known subtypes. 34 of the PCR amplified Among the 34 HIV 1 strains that were characterized DNA fragments of the reactive ELISA and western by sequencing, 19 (55.9%) were subtype G, 9 (26.5%) blot confirmed HIV 1 samples were run at the APIN belonged to CRF_02 A/G , 3 (8.8%) belonged to Laboratory, Harvard University, Department of CRF_06 cpx while 1 (2.9%) each were identified as Immunology and Infectious Diseases, Boston, U.S.A. subtype C, CRF_01 A/E and CRF_09 cpx respectively for the identification of different subtypes by direct (Table 2). sequencing. Phylogenetic analysis was performed by first aligning the sequences obtained with reference Among the 19 subtype G, 9 (47.4%) were obtained sequences. from FSWs who hailed from Edo and Delta states in the South south zone of Nigeria while 3 (15.3%) were Data Analysis from Benue state in the middle belt zone. The only Data was analyzed using SPSS for widows’ version subtypes C identified was from FSW who hailed from 12.0. Association between groups was performed Togo. (Table 3).The youngest (15years) of the using the chi square test for categorical variables and HIV infected FSWs characterized as subtype G has student t test for continuous variables. Logistic been involved in sex trade for less than one year regression was used to adjust for potential while 11 (57.9%) of them have been active in the cofounders. Analysis of variance was calculated and business between one to five years. Also out of the 9 statistical tests were carried out at 5 % significance characterized as CRF_02A/G, 5 (55.6%) have been level active in the trade between 1 5 years. This suggests that there is probability that these infections occurred very recently(P < 0.05).
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Clinical presentations that have been found to be and puritus vulvae (p<0.05). The only clinical significantly associated with HIV 1 subtype G and manifestation noticed with FSW with subtype C CRF_02A/G were weight loss , episodes of recurrent who hailed from Togo was recurrent genital herpes. diarrheas, skin lesions recurrent vaginal discharge
TABLE 1: DEMOGRAPHIC CHARACTERISTICS & REPRODUCTIVE HEALTH HISTORY OF THE COMMERCIAL FEMALE SEX WORKERS
CHARACTERISTICS FREQUENCY PERCENTAGE HIV POSITIVE PERCENTAGE N=250 % N= 64 %
(a) Age < 20 32 12.8 4 6.2 20 24 101 40.4 28 43.8 25 29 56 22.4 17 26.5 30 39 45 18.0 12 18.8 40 49 13 5.2 2 3.1 > 50 3 1.2 1 1.6 (b) Marital Status Single 174 69.6 41 64.1 Married 30 12.0 8 12.5 Divorced 37 14.8 13 20.3 Separated 7 2.8 2 3.1 Widowed 2 0.8 0 0.0 (c) States/Zones/Nationality Edo/Delta 148 59.2 32 50.0 South East 41 16.4 13 20.3 South West 32 12.8 5 7.8 Middle Belt 16 6.4 8 12.5 South South 8 3.2 3 4.7 Other National 4 1.6 3 4.7 (d) Level of Education No formal 15 6.0 3 4.7 Primary education 87 34.8 22 34.4 Secondary 142 56.8 36 56.2 Post secondary 6 2.4 3 4.7 (e ) Age of First Sexual exposure (Yrs) <11 3 1.6 1 1.6 11 15 66 33.2 17 26.5 16 20 109 60.0 41 64.1 >20 8 5.2 5 7.8 (f) Duration of Work as CSWs (Yrs) 2 1.1 2 3.1 <1 112 60.2 37 57.8 1 5 54 29.0 19 29.7 6 10 15 8.1 5 7.8 11 15 3 1.6 1 1.6 16 20 ( g) Number of sexual partners / 119 64.0 41 64.1 day 60 32.2 22 34.4 <5 7 3.8 1 1.5 6 10 186 100.0 64 100.0 >10 Total
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TABLE 2: DISTRIBUTION OF HIV 1 SUBTYPES AMONG FSWS
Subtypes Frequency Percentage % C 1 2.9 G 19 55.9 CRF_01 1 2.9 CRF_02 9 26.5 CRF_06 3 8.8 CRF_09 4 8.8 CRF_09 1 2.8 Total 34 100
TABLE 3: DISTRIBUTION OF HIV 1 SUBTYPES BY AGE AND NIGERIAN ZONES OF ORIGIN
C G CRF01 CRF02 CRF06 CRF09 AGE <20 2(10.6%) 1(11.1%) 20 29 1(100%) 14(73.5%) 1(100%) 4(44.4%) 3(100%) 30 39 1(5.3%) 3(33.3%) 1(100%) 40 49 1(5.3%) 1(11.1%)
50 59 1(5.3%)
Zones of Origin C CRF01 CRF02 CRF06 CRF09
South East 5(26.3%) 1(100%) 1(100%) South West 1(5.3%)
Middle Belt 3(15.7%) 9(100.0%) 3(100%)
South South 10(53.0%)
Other Nationality 1(100%) – Togo
DISCUSSION This study showed that most of the sex workers In Africa the highest sexually transmitted infections (75.6%) were below 30years of age with 62.8% in the (STIs) and HIV infection has been reported among 20 29 years age group while 1.2% were over 50years Female sex workers (FSWs) who are at increasing risk of age. This is consistent with the finding of Bakare et of acquiring and transmitting STIs and Human al(9) that reported 63.3% in 20 29years age group Immunodeficiency Virus (HIV) (17). The AIDS while Umar and other researchers ( 23 24) reported epidemic has exacted a terrible toll in term of life and that younger women in the age group of 20 29 years decreased quality of life worldwide (1). were the ones mostly engaged in sex work. This is not Women, especially young ones, generally lack the a surprise as there is usually higher demand for social power to set the terms for sexual relationship. younger women by men who patronize sex workers. These women live in poverty and are often coerced to trade sex for support and may be forced into The prevalence rate of HIV 1 was 25.6%. There was prostitution. A large member of unmarried, no sex worker with only HIV 2 infection while 2.8% unemployed female brothel based sex workers are all had dual infections of HIV 1 and HIV 2. This over Ibadan and they cater for the sexual needs of the prevalence was however lower than 76.0% reported male populations, mostly of the low income group in studies carried out by Egar et al in Jos (25) and asking for payment in cash in exchange for a short 34.5% by Bakare et al(9,26) in Ibadan. This finding sexual relationship (8). might not be unconnected with the fact that FSWs in their mid to late thirties of age have died of undisclosed illness suspected to be AIDS. Majority of
5 the younger women who are now being introduced recorded in this study was consistent with other were having protected sexual intercourse with studies reported in selected populations in other condoms. African countries (23, 24). Human Immunodeficiency virus type 2 (HIV 2) is less pathogenic than HIV 1. Lower HIV prevalence Among HIV 1 circulating recombinants forms, CRF The genomic diversity of HIV 1 has been a 02 was the predominant and accounted for 26.5% of continuous challenge for the development of the HIV 1 strains identified in this study. This CRF diagnostic test, anti retroviral therapies and the often designated as “1bNG”, the name given to the development of a preventive medicine. first sample described from Ibadan, Nigeria (37). This is contrary to the earlier study(30) where 18.9% of the Among 34 HIV 1 strains characterized, more than half strains belonged to subtype A. Many of the genomes (55.9%) were subtype G, 1(2.9%) belonged to subtype within the 1bNG CRF cluster may have been C. CRF02 which is the most predominant erroneously classified as subtype A. This subtype has recombinant form constitutes about 26.5% of the HIV been responsible for regional epidemics along the strains of the infected FSWs. 3(8.8%) were identified coast of West and East Africa (38). as CRF 06 while others CRF 01 and CRF 09 constitutes Detection of the CRF 06 cpx a complex recombinants 2.9% each respectively. This finding is similar to between Subtypes A, G, J, and K which constituted earlier studies of Abimiku et al ( 28) that reported the 8.8% in this study was in agreement with the findings presence of subtype G from Jos, Nigeria. It is however of Montavon et al ( 39). However, this was the first different with other studies where subtype G time this complex recombinant form has been isolated infections have been described in diverse locations in Nigeria. from East to West Africa, though rarely as a major CRF01 AE which has been responsible for a large proportion of the infection (29) number of infections in Thailand(40) and other South east Asian countries (40) was detected in this study, In different locations in Africa like Mali, Republic of though the prevalence was low (2.9%). Benin, Nigeria and Democratic Republic of Congo, Information about recombinant CRF 09 cpx is very subtype G rarely account for more than 20% all HIV 1 limited (13). Its detection in this study would infections. This is the first time that the prevalence of however form a baseline data for further studies in HIV 1 subtype G is exceeding 50% of the HIV strains this part of the country. The youngest of the HIV identified. Contrary to the study conducted in Nigeria infected FSWs with sexual activity of less than a year by Odaibo et al (30), a heterogeneous distribution of had HIV 1 Subtype G while 55.9% of the FSWs with at least 5 subtypes were observed with subtype C subtype G have been active in the commercial sex being the most predominant in the south western part work between 1 5years. 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